PROJECT SUMMARY/ABSTRACT Passive immunization with broadly-neutralizing antibodies (bnAbs) against HIV-1 offer promise for prevention and even treatment of HIV-1 infection. With the advent of new technologies in recent years, numerous ?broadly neutralizing? antibodies (bnAbs) against HIV-1 have been isolated. While bnAbs appear to be escaped and have little impact on controlling infection in their originating hosts, they have shown promise given as passive immunization, controlling viral replication in infected persons. Bi-specific bnAbs have been shown to be a method to combine bnAbs for synergy. Hybrid bnAbs have been produced by pairing the heavy and light chain of one bnAb with those of another. Proof-of-concept has shown that these can be synergistic combinations of bnAbs. A downside, however, is that these molecules have only a single binding site of each specificity. We propose to utilize a different approach to create bi-specific antibodies. Quadri-valent antibodies can be produced by adding single chain antibody domains to the end of the heavy chain of another antibody. This produces two binding sites for each specificity. Additionally, it allows greater flexibility between the two binding specificities. In this project, we will apply this technology to producing novel quadri-valent chimeric bnAbs. We hypothesize that the additional binding sites and greater flexibility will allow more opportunities for synergy. Specifically, we plan: Aim 1: To create a panel of quadri-valent bi-specific bnAb constructs with modifications that further enhance inter-binding site flexibility, and Aim 2: To test promising constructs for potency and breadth of antiviral activity against HIV-1.